The field of the invention is regulators of enzymes involved in cellular apoptosis.
One of the key regulatory steps for apoptosis is the activation of caspases, leading to the characteristic morphological changes associated with apoptotic cells including chromatin condensation, DNA fragmentation into nucleosomal fragments, nuclear membrane break down, externalization of phosphotidylserine and formation of apoptotic bodies that are readily phagocytosed (Liu et al., 1997 Cell 89, 175-184; Enari et al., 1998 Nature 391, 43-50; Sahara, et al., 1999 Nature 401, 168-173; Lazebnik, et al., 1995 Proc. Natl. Acad. Sci. USA 92, 9042-9046; Martin et al., 1996 J. Biol. Chem. 271, 28753-28756; Zhang et al., 1999 J. Cell Biol. 145, 99-108).
One major apoptotic caspase activation cascade is triggered by cytochrome c, a protein that normally functions in the electron transfer chain in mitochondria (Liu et al., 1996 Cell 86, 147-157). In living cells, holocytochrome c exists exclusively in the intermembrane space of mitochondria, and is therefore sequestered away from its deadly cytosolic partner, Apaf-1 (Zou et al., 1997 Cell 90, 405-413). Upon receiving apoptotic stimuli, such as serum deprivation, activation of cell surface death receptors, and excessive damage of DNA, the outer membrane of mitochondria becomes permeable to cytochrome c (Reviewed by Reed, 1997 Cell 91, 559-562). Once released to cytosol, cytochrome c binds to Apaf-1 with 2:1 stoichiometry and forms an oligomeric Apaf-1/cytochrome c complex in the presence of dATP or ATP (Purring et al., 1999 J. American Chem. Soc. 121, 7435-7436; Zou et al., 1999 J. Biol. Chem. 274, 11549-11556). This oligomerized Apaf-1/cytochrome c complex then recruits and activates the apical caspase of this pathway, procaspase-9 (Li et al., 1997 Cell 91, 479-489; Zou et al., 1999). Caspase-9 in turn activates downstream caspases such as caspase-3, -6 and -7 that constitute the major caspase activity in an apoptotic cell (Li et al., 1997; Srinivasa et al., 1998 Mol. Cell 1, 949-957; Faleiro et al., 1997 EMBO J; 16, 2271-2281).
Here we disclose the identification, purification, molecular cloning, and characterization of a novel factor that promotes cytochrome c/Apaf-1-dependent caspase activation. Like cytochrome c, this protein is normally located in mitochondria and released into cytosol when cells undergo apoptosis. We named this protein Smac, for the second mitochondria-derived activator of caspase, after cytochrome c. Addition of Smac to cytosolic extracts causes robust caspase activation in these extracts even without the addition of dATP. Smac also allows caspase activation in the presence of physiological levels of potassium salt.
The invention provides methods and compositions relating to polypeptide regulators (activators and inhibitors) of enzymes involved in cellular apoptosis, particularly caspases In a particular aspect, the invention provides polypeptide and polynucleotide sequences diagnostic of caspase activators. These sequences and polypeptides and polynucleotides embodying these sequences find a wide variety of diagnostic and therapeutic applications involving detecting and/or modulating expression and/or function of activators or caspases or genes or transcripts encoding such activators. In more particular aspects, the invention provides genetic and immuno probes specific to activators of caspases.
Since undesirable activation or inactivation of apoptosis has been associated with many human diseases such as cancer, autoimmune disease and neurodegenerative diseases, the disclosed caspase regulatory polypeptides and polynucleotides provide both drug targets and regulators to promote or inhibit apoptosis. Also, since disclosed native Smac proteins naturally translocate from mitochondria to cytosol during apoptosis, Smac proteins can be used as diagnostic markers for apoptosis during normal or disease stages, e.g. using labeled Smac proteins such as fusion proteins or using detectable Smac-specific binding agents.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean one or more, the term xe2x80x9corxe2x80x9d means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones described herein.
The subject polypeptide sequences find a wide variety of applications. In one embodiment, the subject sequences are used to synthesize polypeptides which in turn provide a number of applications, including use in proteomic microarrays (e.g. Silzel J W, et al. Clin Chem 1998 September;44(9):2036-43), models for rationale drug design, immunogens for antibody elicitation, etc. The polypeptide sequences are also used to specifically detect sequences comprising SEQ ID NO:2, or fragments thereof, particularly at least one of SEQ ID NO:2, residues 1-78 or SEQ ID NO:2, residues 176-239, or fragments thereof, or polypeptides comprising such sequences. Any convenient sequence detection method may be used, including computational methods for direct sequence detection (e.g. BLAST-type algorithms, alignments, etc.) and physical methods for inferential sequence detection of polymers (e.g. mass spectroscopy, etc.).
In addition to direct synthesis, the subject polypeptides can also be expressed in cell and cell-free systems (e.g. Jermutus L, et al., Curr Opin Biotechnol. 1998 October;9(5):534-48) from encoding polynucleotides, such as the corresponding parent polynucleotides or naturally-encoding polynucleotides isolated with degenerate oligonucleotide primers and probes generated from the subject polypeptide sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.) or polynucleotides optimized for selected expression systems made by back-translating the subject polypeptides according to computer algorithms (e.g. Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166).
The subject polypeptides include fragments of the recited sequences which have Smac-specific amino acid sequence, binding specificity or function. Preferred fragments comprise at least 8, preferably at least 10, preferably at least 15, more preferably at least 25, more preferably at least 35, most preferably at least 50 consecutive residues of SEQ ID NO:2, particularly of at least one of SEQ ID NO:2, residues 1-78 or SEQ ID NO:2, residues 176-239, and have corresponding-polypeptide-specific antibody binding, elicitation or binding or elicitation inhibitory activity.
Specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, etc. Binding assays encompass any assay where the molecular interaction of a subject polypeptide with a binding target is evaluated. The binding target may be a natural binding target such as a regulating protein or a non-natural binding target such as a specific immune protein such as an antibody, or a specific agent such as those identified in screening assays such as described below. Binding specificity may be assayed by binding equilibrium constants (usually at least about 107 Mxe2x88x921, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921), by caspase activation or apoptosis assays, by the ability of the subject polypeptide to function as negative mutants in expressing cells, to elicit specific antibody in a heterologous host (e.g a rodent or rabbit), etc. In a particular embodiment, the subject polypeptide fragments provide specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freunds complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of specific antibodies is assayed by solid phase immunosorbant assays using immobilized corresponding polypeptide, see, e.g. Table 1.
The subject polypeptides and fragments thereof are isolated or pure: an xe2x80x9cisolatedxe2x80x9d polypeptide is unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, and more preferably at least about 5% by weight of the total polypeptide in a given sample and a pure polypeptide constitutes at least about 90%, and preferably at least about 99% by weight of the total polypeptide in a given sample. The polypeptides may be synthesized, produced by recombinant technology, or purified from cells. A wide variety of molecular and biochemical methods are available for biochemical synthesis, molecular expression and purification of the subject compositions, see e.g. Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY) or that are otherwise known in the art.
The invention provides binding agents specific to the subject polypeptides, methods of identifying and making such agents, and their use. For example, specific binding agents are useful in a variety of diagnostic and industrial applications and include somatically recombined polypeptide receptors like specific antibodies or T-cell antigen receptors (see, e.g. Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory), intracellular binding agents identified with assays such as one-, two- and three-hybrid screens, non-natural intracellular binding agents identified in screens of chemical libraries such as described below, etc. Accordingly, the invention provides complementarity determining region (CDR) sequences and libraries of such sequences.
The subject CDR sequences find a wide variety of applications. In one embodiment, the subject CDR sequences are used to synthesize polypeptides which in turn provide a number of applications, including immuno-microarrays, affinity reagents, etc. In addition to direct synthesis, the subject CDR polypeptides can also be expressed in cell and cell-free systems (e.g. Jermutus L, et al., Curr Opin Biotechnol. 1998 October;9(5):534-48) from encoding polynucleotides, such as the corresponding parent polynucleotides or naturally-encoding polynucleotides isolated with degenerate oligonucleotide primers and probes generated from the subject polypeptide sequences (xe2x80x9cGCGxe2x80x9d software, Genetics Computer Group, Inc, Madison Wis.) or polynucleotides optimized for selected expression systems made by back-translating the subject polypeptides according to computer algorithms (e.g. Holler et al. (1993) Gene 136, 323-328; Martin et al. (1995) Gene 154, 150-166). Generally, the CDR polypeptides are expressed and used as the binding domain of an immunoglobulin or fragment thereof.
The invention provides efficient methods of identifying agents, compounds or lead compounds for agents which modulate the ability of the subject polypeptides to interact with a binding target. A wide variety of assays for binding agents are provided including labeled in vitro protein-protein binding assays, immunoassays, caspase activation assay, cell-based assays such as apoptosis assays, etc. The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. In vitro binding assays employ a mixture of components including the subject polypeptide, which may be part of a fusion product with another peptide or polypeptide, e.g. a tag for detection or anchoring, etc. The assay mixtures comprise a binding target. In a particular embodiment, the binding target is an antibody specific for the polypeptide. While native full-length binding targets may be used, it is frequently preferred to use portions thereof so long as the portion provides binding affinity and avidity to the subject polypeptide conveniently measurable in the assay. The assay mixture also comprises a candidate pharmacological agent. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds and are obtained from a wide variety of sources including libraries of synthetic or natural compounds. A variety of other reagents may also be included in the mixture. These include reagents like, salts, buffers, neutral proteins, e.g. albumin, detergents, protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used. The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the polypeptide specifically binds the binding target, portion or analog with a reference binding affinity. The mixture components can be added in any order that provides for the requisite bindings and incubations may be performed at any temperature which facilitates optimal binding. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening. After incubation, the agent-biased binding between the polypeptide and one or more binding targets is detected by any convenient way. A variety of methods may be used to detect the label depending on the nature of the label and other assay components, e.g. through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc. A difference in the binding affinity of the polypeptide to the target in the absence of the agent as compared with the binding affinity in the presence of the agent indicates that the agent modulates the binding of the polypeptide to the binding target. A difference, as used herein, is statistically significant and preferably represents at least a 50%, more preferably at least a 90% difference.
The subject polynucleotide sequences find a wide variety of applications. For example, the polynucleotide sequences are also used to specifically detect Smac sequences, particularly SEQ ID NO:1, its reverse complement or a fragment thereof, preferably at least one of SEQ ID NO:1, nucleotides 1-234 or SEQ ID NO:1, nucleotides 525-720, a reverse complement of either, or a fragment of any thereof, or polynucleotides comprising such sequences. Any convenient sequence detection method may be used. In one embodiment, candidate or unknown sequences are determined and compared with a disclosed sequence to classify the candidate or unknown sequences. For example, an algorithm such as BLAST (e.g. Build sol2.5-x86 01:40:37 Feb. 5, 1998, Copyright (C)1997 Warren R. Gish, using default parameters, Altschul et al., Methods in Enzymology, 215: 403-410 (1997)) may be used to define relatedness to one or more subject sequence diagnostic of Smac-relatedness in computer-based methods.
In another embodiment, the disclosed sequences are used to synthesize and/or are embodied in polynucleotides which in turn provide a number of applications, including microarray-based methodologies, see e.g. Nat Genet 1999 January;21(1 Suppl), entire issue incl. Debouck C, et al. at 48-50; gene expression analysis, see e.g. Carulli J P, et al., J Cell Biochem Suppl 1998;30-31:286-96; drug target discovery and design, see, e.g. Jones D A, et al., Curr Opin Chem Biol 1999 February;3(1):71-6; combinatorial chemistry, see, e.g. Lukas T J, et al., J Med Chem. 1999 March 11;42(5):910-919; ribozymes and therapeutics, see e.g. Rossi J J, Chem Biol 1999 February;6(2):R33-7; mapping; etc. In one embodiment, candidate and/or unknown polynucleotides may be isolated, compared and/or classified (e.g. by relatedness) by hybridization to one or more disclosed polynucleotide, e.g using microarrayed libraries of disclosed polynucleotides. Such polynucleotides may also be used as probes and/or primers to localize, isolate, amplify, etc., natural genes and transcripts. In another embodiment, the disclosed polynucleotides or fragments or libraries of such polynucleotides are transfected into cells for a wide variety of cloning, display, expression, etc. applications, including xe2x80x98nxe2x80x99-hybrid systems, see, e.g. Vidal M, et al., 1999, Nucleic Acids Res. 27(4):919-929 and Proc Natl Acad Sci U S A. 93(19):10315-20 and 10321-6; mapping protein-ligand interactions using whole genome phage display libraries, see e.g. Palzkill T, et al., Gene 1998 October 9;221(1):79-83; DNA-based selection and screening of peptide ligands, see e.g. Bartoli F, et al., Nat Biotechnol 1998 November;16(11):1068-73, etc.
In a particular embodiment, the invention provides microarrays of the disclosed polynucleotides and their uses as described or cited herein. A wide variety of materials and methods are known in the art for arraying polynucleotides at discrete elements of substrates such as glass, silicon, plastics, nylon membranes, etc., including contact deposition, e.g. U.S. Pat. Nos. 5,807,522; 5,770,151, DeRisi J L, et al. Curr Opin Oncol 1999 January;11(1):76-9, etc.; photolithography-based methods, e.g. U.S. Pat Nos. 5,861,242; 5,858,659; 5,856,174; 5,856,101; 5,837,832, Lipshutz R J, et al. Nat Genet 1999 January;21(1 Suppl):20-4, etc.; inkjet dispensing technologies, e.g. Lemmo A V, et al., Curr Opin Biotechnol 1998 December;9(6):615-7; flow path-based methods, e.g. U.S. Pat. No. 5,384,261; dip-pen nanolithography-based methods, e.g. Piner, et al., Science Jan. 29, 1999: 661-663, etc.; etc.
The invention also provides polynucleotides which hybridize to a polynucleotide having a sequence of SEQ ID NO:1, or to its reverse complement. In a particular embodiment, the invention encompasses a recombinant first polynucleotide comprising a sequence at least 36, preferably at least 48, more preferably at least 96 nucleotides in length, the sequence having sequence similarity with a second polynucleotide consisting of SEQ ID NO:1, preferably SEQ ID NO:1, nucleotides 1-234 or SEQ ID NO:1, nucleotides 525-720, or a reverse complement thereof, such that the sequence and second polynucleotide specifically hybridize under a hybridization condition under hybridization condition #1, preferably #2, more preferably #3 and so on to #10, as identified and described in Tables A-C. Thus, for example, if hybridization condition #7 is preferred, then the conditions used for identifying and classifying related or homologous polynucleotides employ hybridization buffer M at a hybridization temperature of 40xc2x0 C., and wash buffer E at awash temperature of 55xc2x0 C. Condition #1 identifies polynucleotides having at least about 50% sequence identity with the target polynucleotide (with % identity calculated as described herein). With each subsequent condition, the stringency is such that the isolated polynucleotide has a sequence identity of at least 5% greater than what would be isolated by using the next lower condition number. Thus, for example, condition #2 identifies polynucleotides having at least about 55% sequence identity with the target polynucleotide, and conditions #9 and #10 identify polynucleotides having at least about 90% and 95% sequence identity, respectively, to the target polynucleotide.
SEQ ID NO:1 is derived from a natural human transcript encoding a natural Smac (see Examples, below). Exemplary higher stringency hybridizing polynucleotides of SEQ ID NO1 (having SEQ ID NO:1 sequence identities of about 95%) are designated SEQ ID NOS:3-5 and exemplary lower stringency hybridizing polynucleotides of SEQ ID NO:1 (having SEQ ID NO:1) sequence identities of about 90%) are designated SEQ ID NOS:6-8 in the Sequence Listing. In situations where it is desired to classify more closely related polynucleotides, the hybridization condition is increased by increments of one, until the desired specificity is obtained. Preferably, each hybridizing polynucleotide has a length that is at least 30%, preferably at least 50%, more preferably at least 70% and most preferably at least 90% of the length of the polynucleotide sequence described herein to which it hybridizes.
In Tables A and B, formamide is expressed as percent (v/v) in a buffered diluent comprising 1xc3x97 to 6xc3x97SSC (1xc3x97SSC is 150 mM NaCl and 15 mM sodium citrate; SSPE may be substituted for SSC, 1xc3x97SSPE is 150 mM NaCl, 10 mM Na H2PO4, and 1.25 mM EDTA, pH7.4). Procedures for polynucleotide hybridizations are well-known in the art (see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995); Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989; Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 6789-6792; and PCT publication WO 99/01466).
The invention also provides fragments of the parent and/or homolog polynucleotides which may be used in the foregoing methods, especially as nucleic acid hybridization probes and replication/amplification primers. These fragments are of length sufficient to specifically the corresponding SEQ ID NO or complement thereof, generally comprising at least 12, preferably at least 24, more preferably at least 36 and most preferably at least 96 contiguous nucleotides of the corresponding SEQ ID NO (see, e.g. Table 2).
The subject polynucleotides include fragments of the recited sequences which have Smac-specific sequence. Preferred fragments comprise at least 34, preferably at least 36, preferably at least 56, more preferably at least 96, more preferably at least 186 consecutive nucleotides of SEQ ID NO:1, particularly of at least one of SEQ ID NO:1, nucleotides 1-234 or SEQ ID NO:1, nucleotides 525-720, a reverse complement of either. The subject polynucleotides and fragments thereof may be joined to other components such as labels or other polynucleotide/polypeptide sequences (i.e. they may be part of larger sequences) and are of synthetic/non-natural sequences and/or are isolated, i.e. unaccompanied by at least some of the material with which it is associated in its natural state, preferably constituting at least about 0.5%, preferably at least about 5% by weight of total nucleic acid present in a given fraction, and usually recombinant, meaning they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome. Recombinant polynucleotides comprising the subject SEQ ID NOs, or fragments thereof, contain such sequence or fragment at a terminus, immediately flanked by (i.e. contiguous with) a sequence other than that which it is joined to on a natural chromosome, or flanked by a native flanking region fewer than 10 kb, preferably fewer than 2 kb, more preferably fewer than 500 bases, most preferably fewer than 100 bases, which is at a terminus or is immediately flanked by a sequence other than that which it is joined to on a natural chromosome. While the nucleic acids are usually RNA or DNA, it is often advantageous to use nucleic acids comprising other bases or nucleotide analogs to provide modified stability, etc.